ACTIVATION OF NUCLEOLAR rRNA GENE TRANSCRIPTION IN LYMPHOCYTES BY TUMOR PROMOTERS: APPLIES STUDIES WITH IN SITU HYBRIDIZATION DETECTED BY FLUORESCENT METHOD

The distribution of rDNA was visualized in interphase nuclei of tumor promoter treated human lymphocytes in comparison with the mltogen Phytoheamagglutinin (PHA) effects by using an in situ hybridization fluorescent method. The procedure involves biotinylated rDNA as the probe and FITC-avidin as detection system. Silver (Ag) staining was used to visualize nucleoli. In the interphase nuclei of most of the nonstimulated control lymphocytes, only one small Ag-stained nucleolus could be seen. The in situ hybridization, however, revealed one to several agglomerations of rDNA fluorescent spots. With tumor promoting herb extract WCE (40 μg/ml) or TPA (60 ng/ ml) treatment, the Interphase nucleoli increased slightly in number with the morphology alteration into larger, reticular or compact granular types. Ag-stained particles also increased in number. The number of the in situ hybridization rDNA fluorescent spots and dots increased markedly and largereticulate formations of numerous rDNA spots were seen. This phenomenum resembles to the changes in PHA stimulated lymphocytes. Statistic analysized data showed signifcant difference between control and drug- treated cells. These results indicate that transcriptionally activated rDNA and amplification of total rDNA was induced by the used tumor promoters.

Clinical role and importance of fluorescence in situ hybridization method in diagnosis of H pylori infection and determination of clarithromycin resistance in H pylori eradication therapy

H pylori is etiologically associated with gastritis, gas-tric and duodenal ulcers, gastric adenocarcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma. Eradicating H pylori may convert rapidly the outcome of related diseases with the use of more accurate diagnostic molecular tests. Indeed some of the tests cannot give the evidence of current infection; H pylori can be detected by noninvasive and invasive methods, the latter requiring an endoscopy. Eradication failure is a big problem in H pylori infection. Recently, clarithromycin resistance in H pylori strains is increasing and eradicati-on therapy of this bacterium is becoming more difficult. Molecular methods have frequently been applied besides phenotypic methods for susceptibility testing to detect clarithromycin resistance due to mutations in the 2143 and 2144 positions of 23S rRNA gene. Fluorescence in situ hybridization (FISH) method on paraffin embedded tissue is a rapid, accurate and cost-effective method for the detection of H pylori infection and to determine clarithromycin resistance within three hours according to the gold standards as a non-culture method. This method can also be applied to fresh biopsy samples and the isolated colonies from a culture of H pylori, detecting both the culturable bacillary forms and the coccoid forms of H pylori, besides the paraffin embedded tissue secti-ons. This technique is helpful for determining the bac-terial density and the results of treatment where clarith-romycin has been widely used in populations to increase the efficacy of the treatment and to clarify the treatment failure in vitro.